Figure 5A Image sequence from confocal time lapse of a neuron that does not extend a nascent axon during nocodazole treatment labelled with a membrane marker and lifeact-Ruby before (-10m) and during (0m to 170m) nocodazole treatment. Protrusions present before nocodazole addition are retracted upon nocodazole treatment (20m). Short, transient non-axonal protrusions are extended during nocodazole treatment. Images are transverse reconstructions from confocal z-stacks. B Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and lifeact-Ruby before (-10m) and during (0h to 170m) nocodazole treatment. Small protrusions present before nocodazole addition (-10m) are retracted upon nocodazole treatment (0m). A nascent axon-like protrusion (long, broad, long-lived) is extended during nocodazole treatment (110m to 170m). Transient non-axonal protrusions are also present. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.C Graph showing duration (minutes) of nascent axon-like protrusions in nocodazole treated cells and non-axonal protrusions in control and nocodazole treated cells. Control nascent axons were not analysed as they do not retract. Bars show mean and standard deviation. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 65.17 minutes, s.d. = 20.33. Control non-axons: n = 40 protrusions from 10 cells from three experiments; mean = 17.15 minutes, s.d. = 8.804. Nocodazole non-axons: n = 41 protrusions from 16 cells from three experiments; mean = 16.182 minutes, s.d. = 7.147. One-way ANOVA with multiple comparisons: P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.9132. D Graph showing length (µm) of nascent axon-like protrusions and non-axonal protrusions in control and nocodazole treated cells. Bars show mean and standard deviation. Control nascent axons were analysed after 60 minutes: n = 18 protrusions from 18 cells; mean = 14.092 µm, s.d. = 5.149. Nocodazole nascent axons: n = 14 protrusions from 14 cells; mean = 9.596 µm, s.d. = 2.371. Control non-axons: n = 40 protrusions from 10 cells; mean = 5.802 µm, s.d. = 2.553. Nocodazole non-axons: n = 41 protrusion from 16 cells; mean = 4.407 µm, s.d. = 2.024. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P = 0.0002; control nascent axon vs control non-axon P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.1485. E Graph showing width (µm) at the base of nascent axon-like protrusions and non-axonal protrusions in control and nocodazole treated cells. Bars show mean and standard deviation. Control nascent axons: n = 19 protrusions from 19 cells from six experiments; mean = 7.806 µm, s.d. = 3.163. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 4.188 µm, s.d. = 1.637. Control non-axons from three experiments: n = 40 protrusions from 10 cells; mean = 1.061 µm, s.d. = 0.652. Nocodazole non-axons from three experiments: n = 41 protrusions from 16 cells; mean = 0.984 µm, s.d. = 0.757. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P < 0.0001; control nascent axon vs control non-axon P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.9958. Data information: *** P < 0.001. **** P < 0.0001.F Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 19 cells from six experiments). Numbers show total count of axons in each quadrant. G Plots showing nascent axon position on the soma of nocodazole treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 14 cells from three experiments). Numbers show total count of axons in each quadrant. H Plots showing the position of non-axonal protrusions on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 40 protrusions from 10 cells from three experiments). Numbers show total count of protrusions in each quadrant. I Plots showing the position of non-axonal protrusions on the soma of nocodazole treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 41 protrusions from 16 cells from three experiments). Numbers show total count of protrusions originating in each quadrant.J Graph showing the ratio of kif5c560-YFP fluorescence intensity in the soma compared to the axon with or without nocodazole. Bars show mean and standard deviation. Control: n = 6 cells from four experiments; mean = 1.81, s.d. = 1.59. Nocodazole: n = 7 cells from four experiments; mean = 0.75, s.d. = 0.19. Data information: *** P < 0.001. **** P < 0.0001.K Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and kif5c560-YFP before (-10m) and during (0h to 144m) nocodazole treatment. A nascent axon-like protrusion is extended during nocodazole treatment (92m to 144m) but kif5c560-YFP does not accumulate there. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.